Molecular and cellular mediators of inflammation play a major role in both the initiation and the progression of stroke. They also participate in the induction of tolerance to ischemia by sublethal preconditioning stresses. We have investigated inflammatory mediators in these three facets of stroke. Inhibition of tumor necrosis factor(TNF) with TNF-binding protein reduces brain infarct volume in middle cerebral artery occlusion (MCAO) models in the rat and mouse. In addition, TNF-binding protein attenuates the progressive impairment of microvascular perfusion that occurs during the early hours of focal brain ischemia. These findings implicate TNF as a mediator of progressive brain damage during acute stroke. Lipopolysaccharide (LPS) pretreatment has been demonstrated to induce tolerance to focal brain ischemia in the MCAO model in the SHR. TNF binding protein blocks this tolerance implicating TNF as the mediator. This form of tolerance also reduces the degree of microcirculatory perfusion impairment in brain. Preconditioning with TNF by intracisternal injection of TNF induces tolerance to ischemia in the Balb/C mouse. In vitro models comprising cellular elements of brain have been established in order to examine the mechanisms involved in the observed in vivo tolerance to ischemia of the brain pretreated either by TNF or by hypoxia/ischemia. Primary cultures of neonatal cortical neurons were subjected to 60 minutes of hypoxia and reoxygenated for various times. Cell death was quantitated using the ethidium homodimer fluorescence exclusion technique. Pretreatment of primary neuronal cultures with short hypoxia (15 minutes) 24 hours prior to 60 minutes of hypoxia, protected neurons against hypoxia (number of dead cells was 9.4% versus 35% in non-pretreated cultures). Pretreatment with TNF (50 ng/ml) or the downstream signaling molecule,ceramide (10 micromolar), also protected cortical neurons against 60 minutes of hypoxia. TNF-preconditioning can induce tolerance to subsequent TNF and hypoxia exposure in BMEC, astrocytes and cortical neurons. Ceramide appears to be involved in the intracellular signaling pathways leading to tolerance of brain cells to ischemia. In in vivo studies, cell-permeable ceramides (C2- and C8-ceramide) have been demonstrated in an adult rat model of focal brain ischemia to reduce infarct volumes. In hypoxic/ischemic insults in neonatal rats, C2-ceramide significantly reduced brain damage, augmented Bcl-2 and Bcl-xl levels, and reduced TUNEL-positive cells. In cortical astrocytes from 2-3 day old Sprague-Dawley rats, preconditioning with TNF-alpha or ceramide to produce tolerance does not inhibit I-kappaB proteolysis, nuclear translocation of NF-kappaB or binding of the p65 subunit of NFkB to its consensus site on DNA. It does, however, prevent p65 phosphorylation and consequently disrupts the association of the coactivator protein, p300/CBP, with that subunit. The result is that expression of proinflammatory genes such as ICAM-1 is inhibited, but expression of cytoprotective genes such as manganese superoxide dismutase continues unabated. In bedside to bench studies, mouse anti-rat ICAM-1 antibody induced an inflammatory state in preclinical models of ischemic stroke that included activation of complement (C3a desArginine), granulocytes (CD11b up-regulation), and endothelium (E- and P-selectin expression). Serial administration of the antibody sensitized rats to produce anti-mouse antibodies and augmented infarct size in a focal brain ischemia model. Similar responses to the mouse anti-human ICAM-1 monoclonal antibody, Enlimomab, may have contributed to the adverse outcome of the Enlimomab acute stroke trial. Immunological tolerization to E-selectin, an adhesion molecule that only becomes expressed on endothelium when a vessel segment becomes activated, has been shown to profoundly reduce ischemic strokes and to eliminate hemorrhages that otherwise occurspontaneously in spontaneously hypertensive and genetically stroke-prone rats (SHR-SP). E-selectin tolerization also can reduce infarct size after MCAO in SHR-SP and this cytoprotection can be adoptively transferred by splenocytes from tolerized animals indicating that the protection is cell-mediated. This novel approach to stroke prevention will be further tested in clinical trials involving preventon of secondary stroke and acute coronary syndromes. Good laboratory practice preclinical toxicology and immunotoxicology studies are in initial stages as prerequisites for obtaining an IND. Ongoing studies in preclinical models examine effects of E-selectin tolerization in vascular dementia, experimental autoimmune encephalomyelitis, delayed vasospasm in subarachnoid hemorrhage, and inflammatory skin disease. A bedside to bench proposal was funded for study of E-selectin tolerization in acute coronary syndromes in collaboration with NINDS Neuroimmunology Branch and NHLBI personnel.